RNA is one kind of nucleic acids, has a different structure from deoxyribonucleic acid (‘DNA’) since it contains ribose instead of deoxyribose and, as a base, uracil instead of thymine. On the basis of structures and functions, the RNA may be classified into a ribosomal RNA (rRNA) to form a ribosome, a messenger RNA (mRNA) having genetic information for synthesis of a protein, which is transferred from DNA, and a transfer RNA (tRNA) to transfer an amino acid corresponding to a codon in the mRNA. Such RNAs may be degraded then synthesized again depending upon the uses and times and an RNase may participate in such processes.
Unlike a deoxyribo-nuclease (‘DNase’), the RNase shows less controlled activity and incurs a problem of easy contamination at any time while collecting a sample until RNA extraction. Also, the RNase may act as a factor of decreasing yield and purity during isolation of pure RNA from a biological sample, therefore, activity thereof must be controlled.
An RNA is generally formed of a single strand and is easily degraded, compared to DNA. For diagnosis of Hepatitis C Virus (‘HCV’), an RNA of the HCV (‘HCV RNA’) in a collected blood sample should be extracted before the HCV RNA is degraded by the RNase, followed by testing to determine the existence of HCV. In this case, if the HCV RNA has been degraded by the RNase in the blood, it may cause significant circumstances to determine a subject who is the owner of the blood sample as a non-infected person, even if he has been infected with the HCV.
Accordingly, it is significantly important to inhibit activity of RNase exposed during extraction of RNAs from a biological sample, however, the most important thing is that the sample is efficiently protected from being exposed to the RNase immediately after the storage of biological samples.
Meanwhile, in order to extract pure RNAs from blood, tissues of animal or plant, culture cells, or the like, two general methods include, for example, use of phenol and use of chaotropic salt.
Phenol well known in the art is a strong organic solvent used to dissolve components of a protein and/or cell, and may degrade the RNase just at the elution of the cells, before the RNase exhibits activity. Therefore, the above material may have an advantage of primarily preventing degradation of RNA. However, phenol also has a disadvantage as a harmful material to the human body and may significantly influence upon further processing if an organic solvent is not completely eliminated.
A chaotropic salt such as a guanidine salt possesses protein modification activity to modify RNase, thus inhibiting the activity of the RNase. The chaotropic salt may also function to attach the RNA to a silica surface, thus having an advantage of enabling easy isolation/purification of RNAs without using any organic solvent.
However, the foregoing methods are to control (inhibit) activity of RNases exposed during isolation of RNAs from a biological sample and, in the case where the RNAs have been already exposed to the RNases while storing the biological sample, cannot attain desired effects even though the activity of the RNases is inhibited during isolation of the RNAs.
An RNase inhibitory enzyme extracted from a human placenta is known to form a 1:1 composite with RNA and be used for non-competitive inhibition, thus being widely employed in the related art (see, Peter Blackburn et. al., JBC, 252:5904-5910 (1977)). If the above RNase inhibitory enzyme extracted from human placenta is treated using a sulfhydryl reagent such as p-hydroxymercury benzoate or N-ethyl maleimide in terms of enzyme features, the composite of RNase and RNase inhibitory enzyme is degraded and inactivated. In order to overcome such disadvantages, excess dithiothreitol (‘DTT’) must be used together with the above composite.
When an animal tissue specimen or the like is stored, a method of preventing degradation of a tissue fragment by RNase wherein the specimen is stored in a 50% or more alcoholic solution or, otherwise, in other solutions such as a polyethylene glycol (PEG) in order to prevent contact between the tissue fragment with moisture, has been reported. However, this method is restricted to experiments such as observation of tissue specimens in a staining process and considered to have limitations in universal or general use in the art (U.S. Pat. No. 7,250,270).
Vanadyl chloride may form a composite with RNase to exhibit effects of suppressing activities of degradation enzymes, however, also have a tendency of inhibiting action of an RNA polymerase. Accordingly, the above compound must be completely eliminated during extraction of the RNA, thus not being preferable (Sambrook et al., 1989).
Based on the foregoing description, currently available compounds for controlling activity of RNase may include: products using RNase inhibitory enzymes isolated from a human placenta as a main material; products containing at least 50% alcohol, which are useful for preventing degradation of RNAs by exposing a sample to alcohol instead of water; products having high concentration salts, which are useful for inhibiting the activity of RNase, or so forth. However, such products have limitation in the kinds of RNases, of which activities are efficiently inhibited by the products, in addition, may encounter a problem of requiring an alternative adjuvant and/or stabilizer added to prolong RNase activity inhibitory ability.